Reverse transcriptase mixtures with improved storage stability

ABSTRACT

Reverse transcriptase mixtures with improved storage stability are provided.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present patent application is a continuation of U.S. patentapplication Ser. No. 13/277,763, filed Oct. 20, 2011, which claimsbenefit of priority to US Provisional Patent Application No. 61/405,961,filed on Oct. 22, 2010, which is incorporated by reference for allpurposes.

BACKGROUND OF THE INVENTION

The detection, analysis, transcription, and amplification of nucleicacids are frequently-used procedures in modern molecular biology. Theapplication of such procedures for RNA analysis can involve theinvestigation of gene expression, diagnosis of infectious agents orgenetic diseases, and the generation of cDNA, to name but a fewapplications. The reverse transcription (“RT”) of RNA thus has manyuses. In some instances, the RT is followed by polymerase chain reactionamplification which can be used for rapid detection and quantificationof RNA. This procedure is often referred to as “RT-PCR”.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for reaction mixtures for reversetranscription of RNA in a sample. In some embodiments, the mixturecomprises: a reverse transcriptase having an optimal pH above 8; and abuffer, wherein the mixture has a pH of between 6-8, lacks template RNA,and the reverse transcriptase maintains essentially all (e.g., at least95% of original) reverse transcriptase activity following incubation ofthe mixture at 50° C. for 30 minutes.

In some embodiments, the reaction mixture comprises sufficientingredients (i.e., such that upon addition of template RNA, underconditions (e.g., temperature) appropriate for the RT enzyme, reversetranscription occurs), except template RNA, for reverse transcription ofRNA.

In some embodiments, the buffer (when measured at a concentration of 0.1M) has a change of no more than 0.027 pH units per degree C. whenbetween 20° and 37° C. In some embodiments, the buffer is selected fromthe group consisting of HEPES, ACES, PIPES, MOPSO, BES, MOPS, TES,TAPSO, POPSO, BICINE, TAPS, and AMPSO.

In some embodiments, the reaction mixture further comprises one or morepolyols having a total concentration of at least 15%. In someembodiments, the polyol is an alcohol having four or more, or five ormore, carbons. In some embodiments, the polyol (e.g., alcohol) isselected from the group consisting of sorbitol and xylitol. In someembodiments, the total concentration of polyols, or total concentrationof at least four carbon alcohols, is at least 25%.

In some embodiments, the total concentration of polyols, or totalconcentration of at least four carbon alcohols, is at least 35%.

In some embodiments, the mixture has a pH less than 8. In someembodiments, the mixture has a pH between 7-8. In some embodiments, themixture has a pH between 7.4-7.8.

In some embodiments, the reverse transcriptase is selected from thegroup consisting of murine leukemia virus (MLV) reverse transcriptase,Avian Myeloblastosis Virus (AMV) reverse transcriptase, RespiratorySyncytial Virus (RSV) reverse transcriptase, Equine Infectious AnemiaVirus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2)reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPTI reverse transcriptase, THERMOSCRIPT reverse transcriptase and MMLVRNase H-reverse transcriptase.

In some embodiments, the mixture further comprises potassium chloride,magnesium, deoxytriphosphate nucleotides, one or more detergents, anoligo dT, an oligonucleotide other than an oligo dT, an RNase inhibitor,or a combination thereof.

In some embodiments, the reaction mixture is sterile.

The present invention also provides for reaction mixtures comprising: areverse transcriptase; and a buffer (when measured at a concentration of0.1 M) that has a change of no more than 0.027 pH units per degree C.when between 20° and 37° C. and when stored within a pH range of 6-8;wherein the mixture lacks template RNA and the reverse transcriptasemaintains essentially all reverse transcriptase activity followingincubation at 50° C. for 30 minutes.

In some embodiments, the reverse transcriptase has an optimal pH above8.

In some embodiments, the reaction mixture comprises sufficientingredients, except template RNA, for reverse transcription of RNA.

In some embodiments, the buffer is selected from the group consisting ofHEPES, ACES, PIPES, MOPSO, BES, MOPS, TES, TAPSO, POPSO, BICINE, TAPS,and AMPSO.

In some embodiments, the reaction mixture has a pH from 6-8. In someembodiments, the mixture has a pH less than 8. In some embodiments, themixture has a pH between 7-8. In some embodiments, the mixture has a pHbetween 7.4-7.8.

In some embodiments, the reverse transcriptase is selected from thegroup consisting of murine leukemia virus (MLV) reverse transcriptaseAvian Myeloblastosis Virus (AMV) reverse transcriptase, RespiratorySyncytial Virus (RSV) reverse transcriptase, Equine Infectious AnemiaVirus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2)reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPTI reverse transcriptase, THERMOSCRIPT reverse transcriptase and MMLVRNase H-reverse transcriptase.

In some embodiments, the mixture further comprises potassium chloride,magnesium, deoxytriphosphate nucleotides, one or more detergents, anoligo dT, an oligonucleotide other than an oligo dT, an RNase inhibitor,a polyol (e.g., wherein the polyol is a four or more, or five or more,carbon alcohol), or a combination thereof.

In some embodiments, the reaction mixture is sterile.

The present invention also provides a mixture comprising: a reversetranscriptase; a buffer; and one or more polyols having a totalconcentration of at least 15%, wherein the mixture lacks template RNAand the reverse transcriptase maintains essentially all reversetranscriptase activity following incubation at 50° C. for 30 minutes. Insome embodiments, the polyol(s) is a four or more, or five or more,carbon alcohol. In some embodiments, the polyol is selected fromsorbitol and xylitol

In some embodiments, sufficient ingredients, except template RNA, forreverse transcription of RNA.

In some embodiments, the buffer (when measured at a concentration of 0.1M) has a change of no more than 0.027 pH units per degree C. when inliquid form between 20° and 37° C. In some embodiments, the buffer isselected from the group consisting of HEPES, ACES, PIPES, MOPSO, BES,MOPS, TES, TAPSO, POPSO, BICINE, TAPS, and AMPSO.

In some embodiments, the total concentration of the polyols (e.g., fouror more, or five or more, carbon alcohol) is at least 25%. In someembodiments, the total concentration of the polyols (e.g., four or moreor five or more carbon alcohol) is at least 35%.

In some embodiments, the reverse transcriptase has an optimal pH above8.

In some embodiments, the mixture has a pH between 6-8. In someembodiments, the mixture has a pH between 7-8. In some embodiments, themixture has a pH less than 8. In some embodiments, the mixture has a pHbetween 7.4-7.8.

In some embodiments, the total concentration of polyols (e.g., four ormore, or five or more, carbon alcohols) is at least 25%. In someembodiments, the total concentration of polyols (e.g., four or more, orfive or more, carbon alcohols) is at least 35%. In some embodiments, thereverse transcriptase is selected from the group consisting of murineleukemia virus (MLV) reverse transcriptase Avian Myeloblastosis Virus(AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV) reversetranscriptase, Equine Infectious Anemia Virus (EIAV) reversetranscriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase,SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reversetranscriptase, THERMOSCRIPT reverse transcriptase and MMLV RNaseH-reverse transcriptase.

In some embodiments, the mixture further comprises potassium chloride,magnesium, deoxytriphosphate nucleotides, one or more detergents, anoligo dT, an oligonucleotide other than an oligo dT, an RNase inhibitor,or a combination thereof.

In some embodiments, the reaction mixture is sterile.

The present invention also provides for methods of reverse transcribingRNA. In some embodiments, the method comprises, contacting a reactionmixture of the invention as described above or elsewhere herein with asample comprising RNA under conditions to allow the reversetranscriptase to reverse transcribe the RNA, thereby producing afirst-strand cDNA.

In some embodiments, the conditions comprise a reverse transcriptiontemperature higher than 42° C.

In some embodiments, the method further comprises forming asecond-strand cDNA.

The present invention also provides for storing an RNA-free ready-to-usereverse transcriptase mixture, the method comprising, storing anRNA-free mixture of the invention as described above or elsewhere hereinat 5° C. or less for at least three days.

In some embodiments, the storing comprises storing the mixture for atleast 14 day.

In some embodiments, the storing occurs at about 4° C.

In some embodiments, the mixture is frozen during the storing.

In some embodiments, the storing occurs at about −20° C. In someembodiments, the storing occurs at about −80° C.

In some embodiments, following the storing, further comprisingcontacting the reaction mixture with a sample comprising RNA underconditions to allow the reverse transcriptase to reverse transcribe theRNA, thereby producing a first-strand cDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates accelerated stability results from reaction mixturesfrozen and then pre-incubated at 50° C. for 30 minutes prior to RT-qPCRanalysis. Control samples were not pre-incubated.

FIG. 2 illustrates the correlation of high and low temperatureincubations in accelerated stability tests. The y-axis represents astability time measurement in minutes indicating the time at which thereaction mixtures lose performance in RT-qPCR. The measurement is basedon 1 Cq delay from its initial measuring point. If the mix is morestable, then more time has passes before the mixtures lose RT-qPCRperformance. For example, at 288° K, mixtures having pH 7.6, 8.0 and 9.0maintained real time stability of 10080, 2880, and 1440 minutes,respectively, demonstrating lower pH mixtures were more stable. Becausethe graph is linear for each of the mixes, the results show acorrelation in accelerated stability, where the mixes are incubated athigh temperature, compared to the real time stability, when the mixesare stored at low temperature such as 4° C. or −20° C.

FIG. 3 illustrates results of RT qPCR for reaction mixtures having HEPESat different pH levels as well as a comparison to a mixture having Trisbuffer.

FIG. 4 illustrates results of accelerated stability tests of reactionmixtures containing various concentrations of xylitol or sorbitol.

FIG. 5 illustrates real-time stability of mixes up to 21 months

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

A reverse transcription (RT) reaction mixture formulation has beendiscovered that can be used in a “ready-to-use” format (i.e., containingreagents sufficient for an RT reaction except the template RNA), hasimproved storage stability, and allows for increased temperature of theRT reaction if desired.

Improved storage of the reaction mixtures described herein can bemanifested, for example, in the ability of the mixtures to be frozen,thawed and then used in an RT reaction with essentially no change in thequantity of cDNA generated. Improved storage stability has been achievedin part by reduction of the pH of the reaction mixture from the standardpH˜8.3 to a pH below 8 (e.g., pH 6-8). Many reverse transcriptaseenzymes commonly used for generation of cDNA have an optimal pH (i.e.,the pH at which activity is optimal) higher than 8. Therefore,historically reverse transcriptase enzymes have been used and stored ata pH higher than 8.

It has been further discovered that the inclusion of a buffer thatexhibits minimal change in buffer capacity over changing temperaturewithin the relevant temperature range for RT reactions and storage(e.g., in liquid samples between −20 and 50° C.) allows for improvedstability of the reverse transcriptase, especially when transitioningbetween storage at colder temperatures (e.g., less than 4° C.) and useat warmer temperatures (e.g., 37° C. and higher).

Stability can be further increased by the inclusion of a relatively highconcentration (at least 15%) of a polyol in the reaction mixture. Whileit is believed other polyols (for example, alcohols having four or moreor five or more carbons) may also be effective, it has been found thatthe use of sorbitol (a six carbon alcohol), xylitol (a five carbonalcohol), or a combination thereof, further improves the thermalstability of reverse transcriptase in a reaction mixture.

As noted above, one benefit from the improved stability of the reactionmixtures of the invention is an improved ability to store reversetranscriptase mixtures while retaining enzyme activity. Yet anotheradvantage is the improved thermostability of the enzymes, especially ofenzymes whose activity is significantly harmed when held at temperaturesgreater than 42° C. at pH 8.3. It is believed that when a reactionmixture of the invention is combined with template RNA, one can performRT reactions at higher temperature than would be possible with astandard RT reaction mixture. Elevated RT reaction temperatures can beuseful, for example, in reducing the occurrence of RNA secondarystructures than can interfere with the RT reaction.

II. Reaction Mixtures

Stabilized aqueous reaction mixtures comprising reverse transcriptaseare provided. “Stable” enzyme reaction mixtures are reaction mixturesthat, for example, can be stored at temperature below −20° C. forprolonged periods of time (e.g., at least 365, 550, or 730 days).“Stability” can also be demonstrated by performing an acceleratedstability test involving stressing the mixture at 50° C. prior totesting its activity and then comparing the activity of the stressedmixture to a mixture that was not stressed. The reaction mixtures of theinvention have improved stability, for example, compared to, standardcommercial mixtures having a pH greater than 8 and containing Trisbuffer.

The reaction mixtures described herein can comprise componentssufficient for an RT reaction aside from the RNA template (i.e., as a“ready-to-use” formulation). This allows for reduced numbers of stepsand solution transfers by a user thereby reducing the opportunity tointroduce error or inconsistency into one's work. Accordingly, whenprepared for commercial sale, the reaction mixtures will typically besterile, for example, to avoid growth of microorganisms during storageand/or introduction of contamination into the RT reaction.

Any of a variety of reverse transcriptases can be used in the reactionmixtures of the invention. Exemplary reverse transcriptases include butare not limited to murine leukemia virus (MLV) reverse transcriptase,Avian Myeloblastosis Virus (AMV) reverse transcriptase, RespiratorySyncytial Virus (RSV) reverse transcriptase, Equine Infectious AnemiaVirus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2)reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPTI reverse transcriptase, THERMOSCRIPT reverse transcriptase and MMLVRNase H⁻ reverse transcriptases. The concentration of the reversetranscriptase can vary and optimal concentrations can be determinedempirically and depend on the particular reverse transcriptase used. Insome embodiments, the reverse transcriptase is at a concentration ofbetween 10-50 units per microliter. It will be appreciated that theconcentration of reverse transcriptase may differ depending on whetherthe reaction mixture is a “pre-mix” lacking the RNA template or thereaction mixture contains the additional volume of RNA template. In someembodiments, the reverse transcriptase will have an optimal pH activityabove pH 8. In some embodiments, the reverse transcriptase is unstable(loses significant activity, for example, resulting in more than 1 Ct orCq delay in an RT-qPCR of a target RNA) following incubation at pH 8.3for 30 minutes at 50° C.

In some embodiments, the reaction mixtures have a pH of 8 or less, e.g.,from 6 to 8, e.g. 6-7, 7-8, 6-7.5, 6.5-8, 7.5-7.7, 7.4-7.8, 7.6, etc.).In some embodiments, the reaction mixture having a pH within one ofthese ranges lacks template RNA but is otherwise “ready-to-use,” i.e.,such that RT occurs upon addition of template RNA and incubation at theappropriate temperature for the RT enzyme to function. Alternatively,the reaction mixture can comprise template RNA, for example during an RTreaction. Generally, the pH of the reaction mixture will be maintainedin part by the presence of a buffer. While it is believed any buffercompatible with RT reactions can be used, it can be further beneficialto use a buffer that does not significantly change buffering activity asa function of temperature with the range of storage and RT reactions(e.g., when in liquid phase between −20° and 50° C.). This can beadvantageous, for example, when the reaction mixture is stored atrelatively cold temperatures (e.g., 4°, −20°, or −80° C.) prior to use,e.g., at between 37°−50° C. In some embodiments, the buffer (whenmeasured at a concentration of 0.1 M) has a change of no more than 0.01,0.02, 0.025, 0.027, or 0.03 pH units per degree C. when between 20° and37° C. This can be determined, for example, by measuring the pKa of thebuffer at 20° and 37° C. and determining the difference in the pKavalues divided by the number of degrees difference (17 degrees).Exemplary buffers that have a change of no more than 0.027 pH units perdegree C. include, but are not limited to, HEPES((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)), ACES(N-(2-Acetamido)-2-aminoethanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid), MOPSO(3-(N-Morpholino)-2-hydroxypropanesulfonic Acid), BES(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic Acid), MOPS(3-(N-morpholino)propanesulfonic acid), TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), TAPSO(3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic Acid),POPSO (Piperazine-N,N′-bis(2-hydroxypropanesulfonic acid)), BICINE(N,N-bis(2-hydroxyethyl)glycine), TAPS(N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), and AMPSO(N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid).Buffers can be used at concentrations appropriate for RT reactions whenthe reaction mixture is mixed with RNA template. In some embodiments,the buffer concentration ranges from 100 to 500 mM, e.g., about 250 mM.

In some embodiments, the reaction mixture comprises one or more polyol.In some embodiments, the polyol is a four or more carbon alcohol or afive or more carbon alcohol. In some embodiments, the total polyolconcentration (i.e., the sum of the concentration of each polyol in thereaction mixture) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40% or more.A variety of different polyols can be used. In some embodiments, thepolyol is selected from sorbitol, xylitol, or a 4 carbon alcohol, a fivecarbon alcohol, a six carbon alcohol or is a combination thereof. Asshown in FIG. 2, concentrations of at least 25% of sorbitol or xylitolare able to stabilize reverse transcriptase (stressed by heat at 55 Cfor 70 minutes), resulting in less than 1 Cq delay as measured byRT-qPCR when compared to an control sample that was not submitted to thestress heating step. Notably, concentrations of 35% or more polyolapparently completely stabilize the reverse transcriptase.

The reaction mixture can contain any additional component sufficientfor, or that improves, an RT reaction. Exemplary additional componentscan include, but are not limited to, potassium chloride (e.g., 100-500mM, e.g., about 250 mM), magnesium (e.g., as MgSO₄, e.g., 10-50 mM,e.g., about 25 mM), deoxytriphosphate nucleotides (e.g., dATP, dCTP,dGTP, dTTP, or analogs thereof), one or more detergents (e.g., Tween20,Triton X-100, NP40, etc., e.g., at 0.1-0.1%, e.g., about 0.05%), anoligo dT (e.g., dT12, dT20, or combinations thereof), an oligonucleotideother than an oligo dT (e.g., a mix of random sequences of a specifiedlength (e.g., a hexamer) or one or more oligonucleotides specific for anRNA sequence of interest), an RNase inhibitor (e.g., Rnasin, RNaseA,etc.), or a combination thereof. In some embodiments, the reactionmixtures comprise a reducing agent. In some embodiments, the reactionmixtures do not comprise a reducing agent or at least does not comprisea sufficient amount of a reducing agent to significantly affectstability of the reverse transcriptase.

Generally, ready-to-use mixtures will contain most or all of theabove-described ingredients. For example, in some embodiments, thereaction mixtures comprise, consist of, or consist essentially of, areverse transcriptase (e.g., having a optimal pH above 8); a buffer (forexample, HEPES, ACES, PIPES, MOPSO, BES, MOPS, TES, TAPSO, POPSO,BICINE, TAPS, or AMPSO); potassium chloride; magnesium;deoxytriphosphate nucleotides; one or more detergents; an oligo dT; anoligonucleotide other than an oligo dT; and optionally an RNaseinhibitor and/or a di-cation chelator (e.g., EDTA). In some embodiments,the reaction mixtures comprise a reducing agent. In some embodiments,the reaction mixtures do not comprise a reducing agent or at least doesnot comprise a sufficient amount of a reducing agent to significantlyaffect stability of the reverse transcriptase.

The reaction mixtures can be packaged as part of a kit if desired,optionally with written instruction materials. Instruction materials canbe in paper or electronic form, for example. In some embodiments, thekit can further comprise control samples (e.g., negative and/or positivecontrols). In some embodiments, the kit comprises a DNA polymerase,e.g., a thermostable polymerase capable of performing a PCR reaction ina thermocycler.

III. Reverse Transcription Reactions

Reverse transcription (RT) is an amplification method that copies RNAinto DNA. RT reactions can be performed with reaction mixtures asdescribed herein. For example, the invention provides for reversetranscribing one or more RNA (including for example, all RNA in a cell,e.g., to make a cDNA library) under conditions to allow for reversetranscription and generation of a first and optionally second strandcDNA. The RT reaction can be primed with a random primer, an oligo dT,or an RNA-specific primer. Components and conditions for RT reactionsare generally known.

If desired, the reactions can further comprise RT-PCR. Standardtechniques for performing PCR assays are known in the art (PCRTechnology: Principles and Applications for DNA Amplification (Erlich,ed., 1989); PCR Protocols: A Guide to Methods and Applications (Innis,Gelfland, Sninsky, &, White, eds., 1990); Mattila et al., Nucleic AcidsRes. 19: 4967 (1991); Eckert & Kunkel, PCR Methods and Applications 1:17 (1991); Wallace et al., Ligase Chain Reaction, in Technologies forDetection of DNA Damage and Mutations, pp. 307-322 (Pfiefer, ed.,1996)). RT and PCR reactions are often used in the same assay and arereferred to as RT-PCR. RT-PCR combines reverse transcription of RNA intoDNA and subsequent DNA amplification reactions in a single reaction.Optimal reverse transcription, hybridization, and amplificationconditions will vary depending upon the sequence composition andlength(s) of the primers and target(s) employed, and the experimentalmethod selected by the practitioner. Various guidelines may be used toselect appropriate primer sequences and hybridization conditions (see,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.)(1989); Ausubel, F. M. et al., eds. (1999) Short Protocols in MolecularBiology, 4th edition, John Wiley & Sons); Ausubel, F. M. et al., eds.(1999-2010) Current Protocols in Molecular Biology, John Wiley & Sons).

The practice of the present invention can employ conventional methods ofchemistry, biochemistry, molecular biology, cell biology, genetics,immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Gennaro, A.R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., MackPublishing Co.; Hardman, J. G., Limbird, L. E., and Gilman, A. G., eds.(2001) The Pharmacological Basis of Therapeutics, 10th ed., McGraw-HillCo.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press,Inc.; Weir, D. M., and Blackwell, C. C., eds. (1986) Handbook ofExperimental Immunology, Vols. I-IV, Blackwell Scientific Publications;Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual,2nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al., eds. (1999-2010) Current Protocols in Molecular Biology,John Wiley & Sons; Ream et al., eds. (1998) Molecular BiologyTechniques: An Intensive Laboratory Course, Academic Press; Newton, C.R., and Graham, A., eds. (1997) PCR (Introduction to BiotechniquesSeries), 2nd ed., Springer Verlag; Sambrook et al., Molecular Cloning: ALaboratory Manual (2nd ed.) (1989).

An advantage of the reaction mixtures of the invention is that thereaction mixtures allow for use of a reverse transcriptase in an RTreaction at a higher temperature than would otherwise be possible. Thus,for example, in embodiments in which 37° or 42° C. would be optimal fora particular reverse transcriptase, the reaction mixtures of theinvention, and in particular reaction mixtures having a pH below 8,allow for the reverse transcriptase to perform an RT reaction above itsoptimal (e.g., 37° or 42° C.) temperature. Thus, in some embodiments, anon-thermostable reverse transcriptase is stored in a ready-to-usereaction mixture of the invention and then used to reverse transcribeone or more RNA at a temperature greater than 42° C., for example,between 43°-55°, 45°-550, 45°-50° C., etc. Higher temperature RTreactions are particularly helpful in situations where the template RNAforms secondary structure at normal RT temperatures (e.g., 37° or 42°C.) that partially or completely inhibit reverse transcription.

IV. Storage

One advantage of the reaction mixtures of the invention is that theyretain essentially all of their activity following storage at lowtemperatures (such as at 4 or −20° C.) or as demonstrated by acceleratedstability tests, i.e., when the mixture are submitted to relatively hightemperature (e.g., 50° or 55° C.) for a period of time. As shown in FIG.1, illustrate a pattern: mixtures having a pH more than 8 were not asstable as mixtures having a pH less than 8 after storage for 2 daysregardless of whether the mixtures were preheated or not. However thepreheated samples show the pattern more clearly. Reaction mixtureshaving a pH of 8 and below did not significantly lose activity followingthe heat treatment. Similarly, FIG. 4 illustrates the improved stabilityof reverse transcriptase reaction mixtures containing variousconcentrations of polyols and demonstrates that increasingconcentrations of these polyols stabilize the reverse transcriptase inaccelerated stability tests.

Reaction mixtures of the invention, including but not limited toready-to-use formulations, can be stored in aqueous form, e.g., frozenor liquid. In some embodiments, the reaction mixtures are stored for atleast 1, 2, 5, 10, 20, 30 days or more without significantly losingreverse transcriptase activity. Reaction mixtures can be stored inliquid or frozen state depending on temperature and composition of themixture, with higher concentrations of polyol (e.g. alcohols with fouror more, or five or more, carbons) for example, allowing for thereaction mixture to remain in liquid form below 0° C., e.g., in someembodiments remaining liquid at around −20° C. In some embodiments, thereaction mixtures are stored at about 4 C, −20 C, or −80 C.

EXAMPLES Example 1 Lower pH Improves Stability

Reaction mixtures comprising MLV reverse transcriptase and havingvarious pH levels were stored frozen and in liquid state at −20 C bytreating or not treating the mixes with a quick freeze on dry ice priorto storage. The reaction mixtures consisted of 250 mM Tris at pH 7.4,7.5, 7.8, 8.0, 8.3, 8.7, and 9.0, 25 mM Mg₂SO₄, 250 mM KCl, 2.5 mMdNTPs, 5 μl of RTase, 1 mM EDTA, 28.4 μM of Oligo Primers and 12.5%Glycerol. After two days of storage at −20 C, sets of the mixtures,frozen or not frozen, were pre-incubated at 50° C. for 30 minutes priorto usage in RT-qPCR with 100 ng/rxn of Hela RNA to detect 18s RNA.Control samples were treated identically but were not pre-incubated at50° C. The thermocycling condition for the RT step was 25° C. 5 mins,42° C. 30 mins and 85° C. 5 mins and for the qPCR step was 95° C. 5mins, 95° C. 10 sec 60° C. 30 sec×45 cycles plus a melt curve analysis.

The results of the experiment showed that both freezing and lowering thepH to 8 or below has an additive effect on storage stability. Moreover,lowering the pH significantly improves stability of both frozen andunfrozen mixtures. See, FIG. 1. Stressing the mixtures at an elevatedtemperature for a period of time prior to RT-qPCR resulted in a loss inperformance when pH was increased from 8.0 to 8.5 between mixes thatwere frozen or liquid. Similarly, there was a loss of RT-qPCR activitywhen pH was raised to 9.0 with the frozen mixtures. By lowering the pHbelow 8.0, the storage stability of the mixtures were stabilized in thefrozen or liquid mixtures regardless of whether the samples werepre-incubated at 50° C.

Example 2 Pre-Incubation at High Temperature Correlates to LowTemperature Storage Stability

To illustrate that the accelerated stability test involvingpre-incubation at 50° C. correlated to low temperature stability,several mixtures where incubated at various time points at varioustemperatures. Three different mixtures were stored at −20 C with thefollowing compositions: 250 mM Tris at pH 9.0, 8.0 or 7.6, 25 mM Mg₂SO₄,250 mM KCl, 2.5 mM dNTPs, 5 μl of RTase, 1 mM EDTA, 28.4 μM of OligoPrimers and 18% Glycerol. The mixtures were incubated at various timepoints and various temperatures prior to RT-qPCR with 100 ng/rxn of HelaRNA to detect 18s RNA. The thermocycling condition for the RT step was25° C. 5 mins, 42° C. 30 mins and 85° C. 5 mins and the qPCR step was95° C. 5 mins, 95° C. 10 sec 60° C. 30 sec×45 cycles plus a melt curveanalysis.

FIG. 2 shows a correlation graph comparing the stability of the mixesincubated at high temperature and low temperature of time. As theincubation temperature increased, less time was needed to stress the mixbefore RT-qPCR performance was affected. Lowering the pH level of themixture improved stability. For example, the slope of the mix at pH 7.6was steeper compared to the others, demonstrating stability for longerperiods of time at a given temperature. Based on an Arrhenius equation,the results provide an estimated shelf life of 4 years for the pH 7.6sample when stored at −20° C.

Example 3 HEPES Buffer Provides Better Stability

It was also discovered that replacing Tris buffer with HEPES alsoimproved the storage stability of the mixture. For example,HEPES-containing mixtures had improved stability based on incubations of30 days at −20 C and also based on accelerated stability tests. Theformulation tested consisted of 250 mM Tris pH 7.6 or 250 mM HEPES pH7.4, 7.6 or 8.0, 25 mM Mg₂SO₄, 250 mM KCl, 2.5 mM dNTPs, 5 μl of RTase,1 mM EDTA, 28.4 μM of Oligo Primers and 32% Glycerol. After 30 days ofstorage at −20° C., mixtures were pre-incubated at 55° C. for 90 minutesprior to usage in RT-qPCR with 100 ng/rxn of Hela RNA with 18s assay.Control mixtures were tested but were not pre-incubated at 55° C. Thethermocycling condition for the RT step was 25° C. 5 mins, 42° C. 30mins and 85° C. 5 mins and for the qPCR step was 95° C. 5 mins, 95° C.10 sec 60° C. 30 sec×45 cycles plus a melt curve analysis. The controlin FIG. 3 represents a freshly made mix prior to RT-qPCR with nopre-incubation step.

These data demonstrate that lowering the pH improves stability of themixes in accelerated stability tests. In addition, based on theaccelerated stability after 30 days, the HEPES pH 7.6 compared to theTris pH showed better accelerated stability. By switching to HEPES, thesmall change in pH in respect to temperature helped stabilize the mixstored at −20 C.

Example 4 Increasing in Sorbitol and Xylitol Concentration ImprovesStability

FIG. 4 shows data from experiments in which the mixtures have increasingamounts of sorbitol and xylitol. Mixtures were treated with or (control)without a pre-incubation step for 55° C. for 70 minutes prior toRT-qPCR. Each mixture consisted of 250 mM HEPES pH 7.6, 25 mM Mg₂SO₄,250 mM KCl, 2.5 mM dNTPs, 5 μl of RTase, 1 mM ETDA, 40 μM Oligo Primers,and 15%-35% xylitol or 15%-25% sorbitol. The mixtures were treated withor without the pre-incubation step, followed by RT-qPCR with 18s primerset, 100 ng/rxn of Hela RNA and iQ SBYR SuperMix (Bio-Rad, Hercules,Calif.). The thermocycling condition for the RT step was 25° C. 5 mins,42° C. 30 mins and 85° C. 5 mins and for the qPCR step was 95° C. 5mins, 95° C. 10 sec 60° C. 30 sec×45 cycles plus a melt curve analysis.The control (“no treatment”) represents a freshly made mix prior toRT-qPCR with no pre-incubation step.

Lowering the pH and increasing the sugar (polyol) of a reaction mixtureimproves the stability of reaction mixtures. The data demonstrates thatincreasing the amount of sorbitol or xylitol from 15% to 35% helpsstabilize the mixtures. As sorbitol or xylitol concentration isincreased, there is less change in RT-qPCR performance between thetreated (pre-incubated) or untreated samples at elevated temperature.Based on the accelerated stability studies, an estimate shelf life of 4years can be expected from mixtures having 25% sorbitol or 35% xylitol.

Example 5 Mixes Shows Real Time Stability for Up to 21 Months

Several mixes, which are identical in composition, were stored at −20°C. for 21, 20 and 12 months and measured functionally by RT-qPCR todetermine the relative stability to a control mix. The comparison ofeach mix shows less than 1 ΔCq difference to the control mix. Eachmixture consist of 250 mM HEPES pH 7.6, 25 mM Mg₂SO₄, 250 mM KCl, 2.5 mMdNTPs, 5 μl of RTase, 1 mM ETDA, 40 μM Oligo Primers, and Polyol. Themixes were used to synthesize cDNA from 100 ng/rxn of Hela RNA with 18sand GAPDH primer sets and followed by qPCR with iQ SBYR SuperMix(Bio-Rad, Hercules, Calif.). The thermocycling condition for the RT stepwas 25° C. 5 mins, 42° C. 30 mins and 85° C. 5 mins and for the qPCRstep was 95° C. 5 mins, 95° C. 10 sec 60° C. 30 sec×45 cycles plus amelt curve analysis. The control represents a freshly made mix inRT-qPCR.

The examples and embodiments described herein are for illustrativepurposes only and that various modifications or changes in light thereofwill be suggested to persons skilled in the art and are to be includedwithin the spirit and purview of this application and scope of theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference in their entirety forall purposes.

What is claimed is:
 1. A sterile aqueous reaction mixture comprisingsufficient ingredients, except template RNA, for reverse transcriptionof RNA, the mixture comprising: a reverse transcriptase having anoptimal pH above 8; deoxytriphosphate nucleotides; and a buffer, whereinthe buffer is selected from the group consisting of Tris, HEPES, ACES,PIPES, MOPSO, BES, MOPS, TES, TAPSO, POPSO, BICINE, TAPS, and AMPSO,wherein said sterile aqueous reaction mixture has a pH of between 6-8and lacks template RNA.
 2. The sterile aqueous reaction mixture of claim1, wherein said buffer, when measured at a concentration of 0.1 M, has achange of no more than 0.027 pH units per degree Celsius when between20° C. and 37° C.
 3. The sterile aqueous reaction mixture of claim 1,further comprising one or more polyols having a total polyolconcentration of at least 15%, wherein said polyol is a four or morecarbon alcohol.
 4. The sterile aqueous reaction mixture of claim 3,wherein said polyol is selected from the group consisting of sorbitoland xylitol.
 5. The sterile aqueous reaction mixture of claim 1, whereinsaid reverse transcriptase is selected from the group consisting ofmurine leukemia virus (ML V) reverse transcriptase, Avian MyeloblastosisVirus (AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV)reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reversetranscriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase,SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reversetranscriptase, THERMOSCRIPT reverse transcriptase and MMLV RNase Hreverse transcriptase.
 6. The sterile aqueous reaction mixture of claim1, wherein said sterile aqueous reaction mixture further comprisespotassium chloride, magnesium, one or more detergents, an oligo dT, anoligonucleotide other than an oligo dT, an RNase inhibitor, or acombination thereof.
 7. The sterile aqueous reaction mixture of claim 1,consisting of said reverse transcriptase, said buffer, potassiumchloride, MgSO4, one or more polyol(s), EDTA, and oligonucleotides. 8.The sterile aqueous reaction mixture of claim 1, wherein said reversetranscriptase is selected from the group consisting of SUPERSCRIPT IIreverse transcriptase, SUPERSCRIPT I reverse transcriptase, THERMOSCRIPTreverse transcriptase and MMLV RNase H-reverse transcriptase.
 9. Thesterile aqueous reaction mixture of claim 1, further comprising one ormore detergents.
 10. The sterile aqueous reaction mixture of claim 1,further comprising an oligo dT oligonucleotide.
 11. The sterile aqueousreaction mixture of claim 1, wherein said buffer is at a concentrationof between 100-500 mM.
 12. A method of storing the sterile aqueousreaction mixture of claim 1, the method comprising storing the sterileaqueous reaction mixture at 5° C. or less for at least three days. 13.The method of claim 12, wherein the storing comprises storing themixture for at least 14 day.
 14. The method of claim 12, wherein thestoring occurs at 4° C.
 15. The method of claim 12, wherein the mixtureis frozen during the storing.
 16. The method of claim 12, wherein,following the storing, further comprising contacting the reactionmixture with a sample comprising RNA under conditions to allow thereverse transcriptase to reverse transcribe the RNA, thereby producing afirst-strand cDNA.